神经管畸形与正常胚胎脑组织miRNAs差异表达分析及其靶mRNA的功能预测

发布时间：2018-03-20 15:13

  本文选题：神经管畸形　切入点：无脑畸形　出处：《中国协和医科大学》2010年博士论文　论文类型：学位论文

【摘要】： 神经管畸形(neural tube defects,NTDs)是由于在胚胎发育过程中,神经管闭合不全所引起的一类先天畸形。NTDs属多因素疾病,该病的发生主要是遗传因素和环境因素及两者相互作用的结果。这种畸形起因于神经管闭合的失败,所以影响神经管发育的因素都可能促使其形成。MicroRNAs (miRNAs)是一类长约22个核苷酸的内源性单链非编码RNA,通过与靶mRNA完全或不完全互补配对,导致靶mRNA降解或抑制其翻译,从而在基因的表达调控中发挥着重要作用。目前研究表明miRNAs在神经干细胞分化和哺乳动物脑发育过程中起重要作用,提示miRNAs可能参与神经管畸形的发生。但以往研究检测的miRNA数量有限,并且国内外尚无miRNA在胚胎脑组织中功能研究的报告。因此有必要深入开展miRNA在NTDs胚胎脑组织中的表达和功能研究,为深入了解NTDs的发病机理提供新的线索。本课题通过对于miRNAs在NTDs与正常胚胎脑组织的差异表达分析和特定miRNAs作用机制的阐释,有助于我们进一步提高对于NTDs发生发展的分子机制的认识。 目的： 建立神经管畸形与正常胚胎脑组织miRNAs的差异表达谱,利用生物信息学手段,预测差异表达显著的miRNAs的靶基因,并进一步分析靶基因的生物学功能及在神经管畸形发生中的作用。为进一步探讨miRNAs在神经管畸形发生机制中的作用提供依据。 方法： 利用1niRCURYTM LNA miRNA表达谱芯片分析无脑畸形残余脑组织和正常胎儿脑组织miRNAs差异表达。利用NCodeTM qRT-PCR kit试剂盒,以U6 snRNA作为内标,对差异表达显著的miRNAs进行验证。利用mirGEN v3数据库预测差异表达显著的9个miRNAs的靶基因,利用Human Protein Reference Database (HPRD)分析靶蛋白的相互作用,建立相互作用网。利用GeneCodis 2.0分析相互作用网所含蛋白质的生物学功能,并以此推断miRNAs在NTDs发生发展中的作用。 结果： 1. miRNA芯片检测发现：实验组与对照相比,有213个miRNAs表达差异显著(两组差异2倍)。niRNAs表达上调2倍以上116个(其中包含1niRPLUS 9个),miRNAs表达下调2倍以上97个(其中包含miRPLUS 2个) 2.依据表达差异显著性和在脑组织中表达的特异性,选择9个miRNAs进行real-time RT-PCR验证。miR-9、miR-212、miR-451、miR-198、miR-126、miR-124和miR-138检测结果与miRNA芯片一致,miR-103/miR-107与miRNA芯片结果相反。表达上调：miR-451、miR-198和miR-126,表达下调：miR-9、miR-212、miR-103、miR-107、miR-124和miR-138。这9个miRNAs在NTDs和正常对照中的表达差异均具有统计学意义(p0.05) 3.经过mirGEN v3数据库分析,881个基因被预测为这9个1niRNAs的靶基因。其中79个基因包含在蛋白质相互作用网中。 4.经GeneCodis 2.0分析,这些蛋白参与基因转录、信号转导、细胞周期、细胞生长、死亡和细胞间信号转导这些重要的生物学过程；发挥蛋白结合、转录因子活化、受体结合及活化、核酸结合、DNA结合和转移酶活化等生物学功能；并且它们涉及肌动蛋白、Jak-STAT和MAPK等与神经发育相关的信号通路。 结论： miRNAs在无脑畸形和正常对照胎儿脑组织中表达谱存在差异,可能参与了无脑畸形发生的分子机制。同时,本研究绘制了靶基因的蛋白质相互作用网,并分析了这些靶蛋白的生物学功能,为深入研究这些miRNAs的调控机制及在NTDs发生发展中的作用提供了基础。
[Abstract]:Neural tube defects (neural tube, defects, NTDs) is due to the process of embryonic development, a kind of neural tube malformation caused by the incomplete closure of.NTDs is a multifactorial disease, the incidence of the disease is genetic and environmental factors and the interaction between the two results. This deformity is attributed to the failure of neural tube closure, so factors affecting the development of the neural tube are likely to promote the formation of.MicroRNAs (miRNAs) is a single stranded endogenous 22 nucleotides encoding non RNA, with target mRNA complete or incomplete complementary, leading to degradation of target mRNA or inhibiting the translation, resulting in the regulation of gene expression plays an important role in the present. Research shows that miRNAs plays an important role in the process of development and differentiation of nerve stem cells in the mammalian brain, suggesting that miRNAs may be involved in the occurrence of neural tube defects. But previous studies detected a limited number of miRNA and And there is no report on miRNA function in embryonic brain tissue. So it is necessary to carry out the expression and function of miRNA in brain tissue in NTDs embryos, and provides new clues to the pathogenesis of in-depth understanding of NTDs. Through the research for the differences in miRNAs NTDs and normal embryonic brain tissue specific expression analysis and miRNAs the mechanism of interpretation, contribute to the improvement of our understanding of the molecular mechanism of the occurrence and development of NTDs.
Objective:
Differential expression of neural tube defects and normal embryonic brain tissue miRNAs established by the method of bioinformatics, gene expression of miRNAs was predicted differences, and further analysis of the biological function of target gene and tube malformation in nerve. In order to further explore the miRNAs incidence of neural tube defect mechanism provides the basis for.
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